重组细粒棘球蚴亲肌肉抗原抗小鼠感染细粒棘球蚴的免疫保护性及其机制研究

发布时间：2018-05-11 08:00

本文选题：细粒棘球蚴 + 重组亲肌肉抗原　；参考：《宁夏医科大学》2010年硕士论文

【摘要】：目的:对构建的纯化重组亲肌肉抗原(rEg.myophilin)进行免疫保护和免疫机制的研究,为包虫病分子疫苗的研制提供新的候选分子。方法:(1)疫苗制备:重组Eg.myophlilin/pET-28a工程菌株的大量表达、纯化及疫苗制备。(2)动物分组方案:选取111只小鼠随即分为A、B对照组和实验组C组,分别对其进行皮下PBS、PBS+佐剂和PBS加佐剂加候选疫苗的注射,之后分别用同样的试剂对其对应组进行两次加强免疫;最后用1500只细粒棘球蚴的原头蚴腹腔攻击感染。(3)免疫保护力计算:(免疫保护力计算公式:减囊率=1-实验组平均包囊数/对照组平均包囊数×100%)。(4)对小鼠进行免疫保护机制的研究,①体液免疫抗体指标的测定:从领取小鼠开始按不同的时间点,分别分批次对小鼠采血,进行血清IgG、IgG1、IgG2a、IgG2b、IgG3和IgE水平的测定;②细胞免疫检测:从领取小鼠即开始按不同的时间点,分别分批次对小鼠取脾脏,并脾细胞刺激培养,监测其细胞因子IFN-γ、IL-4和IL-10的水平。结果:(1) rEg。myophilin能诱导小鼠产生86.11%以上的免疫保护力。(2)采集血清进行ELISA血清抗体IgG、IgG1、IgG2a、IgG2b、IgG3和IgE以及细胞因子IFN-γ、IL-4和IL-10的检测,结果表明:抗体IgG、IgG1、IgG2a以及IgE,和细胞因子IFN-γ、IL-4分别在免疫之后出现了增高,对照组IL-10在攻击感染后出现了明显的增高,而在实验组则没有相应的增高。结论:(1)rEg.myophilin为极具应用潜质的疫苗候选分子。(2)对rEg.myophilin产生的免疫保护性可能机制:①抗体介导的体液免疫,IgG及其亚类IgG1和IgE介导的体液免疫,在抗细粒棘球蚴感染过程中发挥重要作用;②细胞免疫中,细胞因子水平变化显示Th1类免疫应答在试验组免疫保护中占优势作用,有利于机体抵御细粒棘球蚴的感染;③该疫苗候选分子的注入使得免疫抑制机制得到了有效地阻遏,有利于机体的抗寄生虫。④体液免疫和细胞免疫相辅相成,相互促进,表现在:IL-4在IgE亚型类别转换中起到了关键的作用,这一机制可能直接影响着IgE参与的嗜酸性粒细胞相关的抗寄生虫感染。
[Abstract]:Objective: to study the immune protection and immunological mechanism of the purified recombinant muscular antigen rEg.myophilin in order to provide a new candidate for the development of hydatidosis vaccine. Methods 1) Vaccine preparation: recombinant Eg.myophlilin/pET-28a engineering strain was expressed, purified and vaccine preparation. 2) Animal grouping: 111 mice were randomly divided into two groups: control group and experimental group C, the control group was divided into two groups, the control group and the control group, and the control group was divided into two groups, the control group and the control group. They were injected with subcutaneous PBS- PBS adjuvant and PBS plus adjuvant plus candidate vaccine respectively, and then their corresponding groups were immunized twice with the same reagent. At last, the immune protective mechanism of 1500 echinococcus granulosus was studied by calculating the immune protection ability of the mice by using the immune protection power (the formula of the immune protective power: the ratio of reducing the cysts was 1- the average number of the cysts in the experimental group and the average number of the cysts in the control group 脳 100th), and the immune protection mechanism of the mice was studied by using the immune protection ability of 1500 echinococcus granulosus. 1 determination of humoral immune antibody index: blood samples were collected from mice at different time points from receiving mice, and the levels of IgG2G1 and IgE in serum were determined by different time points, and the levels of IgG2G2bG3 and IgE were detected by different time points from the receiving mice, and the blood samples were collected from each group of mice at different time points, and the levels of IgG2bG3 and IgG2bG3 in the sera of the recipient mice were determined at different time points. Spleen was collected from mice in different batches, and spleen cells were stimulated and cultured to monitor the levels of IL-4 and IL-10. Results rEg.myophilin could induce mice to produce more than 86.11% of immuno-protective power.) Serum samples were collected to detect IgG3 and IgE, and cytokine IFN- 纬 IL-4 and IL-10. The results showed that the levels of IgG2a, IgE, and cytokine IFN- 纬 IL-4 were increased after immunization. The IL-10 of the control group was significantly increased after infection, but not in the experimental group. Conclusion the possible mechanism of immune protection of rEg.myophilin induced by 1: 1 antibody against rEg.myophilin and its subclasses of IgG1 and IgE mediated by IgG1 and IgE may be the possible mechanism of immuno-protective mechanism of IgG1. Myophilin is a potential vaccine candidate molecule. In the process of anti-infection of Echinococcus granulosus, the change of cytokine level showed that the immune response of Th1 was dominant in the immune protection of the experimental group, which was beneficial to resist the infection of echinococcus granulosus. (3) the injection of candidate vaccine molecules makes the immunosuppressive mechanism effectively repress, which is beneficial to the body's anti-parasite .4 humoral immunity and cellular immunity, which complement each other and promote each other. This mechanism may directly affect the anti-parasitic infection associated with eosinophilic granulocytes involved in IgE.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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